1. What is the BCA Assay?

The Bicinchoninic Acid (BCA) assay is a colorimetric method for quantifying total protein concentration. It's based on the reduction of Cu²⁺ to Cu⁺ by proteins in an alkaline medium, followed by color development with BCA.

2. How Does the Calculator Work?

The calculator uses the BCA standard curve equation:

Where:

• \( \text{Absorbance} \) — Measured absorbance value (unitless)
• \( C \) — Y-intercept from standard curve (unitless)
• \( m \) — Slope from standard curve (mL/µg)

Explanation: The equation calculates protein concentration based on the linear relationship between absorbance and protein concentration established by the standard curve.

3. Importance of Protein Quantification

Details: Accurate protein quantification is essential for various biological experiments including Western blotting, enzyme assays, and protein purification workflows.

4. Using the Calculator

Tips: Enter the measured absorbance value, the intercept (C) and slope (m) values from your standard curve. Ensure all values are from the same standard curve experiment.

5. Frequently Asked Questions (FAQ)

Q1: What is the typical range for BCA assays?
A: The BCA assay typically works in the range of 20-2000 µg/mL, though this can vary based on the specific protocol and standard curve.

Q2: How often should I create a new standard curve?
A: A new standard curve should be created for each experiment to account for day-to-day variations in reagents and conditions.

Q3: What affects BCA assay accuracy?
A: Factors include sample composition, interfering substances, pH, temperature, and incubation time.

Q4: Can BCA be used with any protein?
A: While BCA works with most proteins, different proteins may produce slightly different color responses due to variations in amino acid composition.

Q5: How should samples be diluted?
A: Samples should be diluted to fall within the linear range of your standard curve, typically requiring preliminary testing.